| Summary: | The protein biomarker measurement has been well-established using ELISA (enzyme-linked immunosorbent assay), which offers good sensitivity and specificity, but remains slow and expensive. Certain clinical conditions, where rapid measurement or immediate confirmation of a biomarker is paramount for treatment, necessitate more rapid analysis. Biosensors offer the prospect of reagent-less, processing-free measurements at the patient’s bedside. Here, we report a platform for biosensing based on chelated Eu<sup>3+</sup> against a range of proteins including biomarkers of cardiac injury (human myoglobin), stroke (glial fibrillary acidic protein (GFAP)), inflammation (C-reactive protein (CRP)) and colorectal cancer (carcinoembryonic antigen (CEA)). The Eu<sup>3+</sup> ions are chelated by modified synthetic binding proteins (Affimers), which offer an alternative targeting strategy to existing antibodies. The fluorescence characteristics of the Eu<sup>3+</sup> complex with modified Affimers against human myoglobin, GFAP, CRP and CEA were measured in human serum using λ<sub>ex</sub> = 395 nm, λ<sub>em</sub> = 590 and 615 nm. The Eu<sup>3+</sup>-Affimer based complex allowed sensitive detection of human myoglobin, GFAP, CRP and CEA proteins as low as 100 fM in (100-fold) diluted human serum samples. The unique dependence on Eu<sup>3+</sup> fluorescence in the visible region (590 and 615 nm) was exploited in this study to allow rapid measurement of the analyte concentration, with measurements in 2 to 3 min. These data demonstrate that the Affimer based Eu<sup>3+</sup> complexes can function as nanobiosensors with potential analytical and diagnostic applications.
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